Dora Beshkova

Carotenoids from Rhodotorula and Phaffia : yeasts of biotechnological importance

Carotenoids represent a group of valuable molecules for the pharmaceutical, chemical, food and feed industries, not only because they can act as vitamin A precursors, but also for their coloring, antioxidant and possible tumor-inhibiting activity. Animals cannot synthesize carotenoids, and these pigments must therefore be added to the feeds of farmed species. The synthesis of different natural commercially important carotenoids (β-carotene, torulene, torularhodin and astaxanthin) by several yeast species belonging to the genera Rhodotorula and Phaffia has led to consider these microorganisms as a potential pigment sources. In this review, we discuss the biosynthesis, factors affecting carotenogenesis in Rhodotorula and Phaffia strains, strategies for improving the production properties of the strains and directions for potential utility of carotenoid-synthesizing yeast as a alternative source of natural carotenoid pigments.

Exopolysaccharides Produced by Lactic Acid Bacteria of Kefir Grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12. and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.

Caroteno-protein and exopolysaccharide production by co- cultures of Rhodotorula glutinis and Lactobacillus helveticus

The lactose-negative yeast Rhodotorula glutinis 22P and the homofermentative lactic acid bacterium Lactobacillus helveticus 12A were cultured together in a cheese whey ultrafiltrate containing 42 g L−1 lactose. The chemical composition of the caroteno-protein has been determined. The carotenoid and protein contents are 248  μ g g−1 dry cells and 48.2% dry weight. Carotenoids produced by Rhodotorula glutinis 22P have been identified as β-carotene 15%, torulene 10%, and torularhodin 69%. After separating the cell mass from the microbial association, the exopolysaccharides synthesized by Rhodotorula glutinis 22P were isolated from the supernatant medium in a yield of 9.2 g L−1. The monosaccharide composition of the synthesized biopolymer was predominantly D-mannose (57.5%).

Effect of Aeration on the Production of Carotenoid Pigments by Rhodotorula rubra-lactobacillus casei Subsp. casei Co-Cultures in Whey Ultrafiltrate

Under intensive aeration (1.3 l/l min) the associated growth of Rhodotorula rubra GED2 and Lactobacillus casei subsp. casei in cheese whey ultrafiltrate (55 g lactose/l) proceeded effectively for both cultures with production of maximum carotenoids (12.4 mg/l culture fluid). For maximum amount of carotenoids synthesized in the cell, the yeast required more intensive aeration than the aeration needed for synthesis of maximum concentration of dry cells. Maximum concentration of carotenoids in the cell (0.49 mg/g dry cells) was registered with air flow rate at 1.3 l/l min, and of dry cells (27.0 g/l) at 1.0 l/l min. An important characteristic of carotenogenesis by Rhodotorula rubra GED2 + Lactobacillus casei subsp. casei was established - the intensive aeration (above 1.0 l/l min) stimulated β-carotene synthesis (60% of total carotenoids).

Bacteriocins from lactic acid bacteria: Microorganisms of potential biotechnological importance for the dairy industry

Bacteriocins are a heterogeneous group of ribosomally synthesized, extracellularly released, bioactive peptides or proteins displaying antimicrobial activity against other bacteria. Over the last two decades, there has been an explosion of basic and applied research on lactic acid bacteria (LAB) bacteriocins, primarily due to their potential application as biopreservatives in food and food products to inhibit the growth of food‐borne bacterial pathogens. Although bacteriocins can be produced in the food matrix during food fermentation (in situ), bacteriocins by LAB can be produced in much higher amounts during in vitro fermentations under optimal physical and chemical conditions. Because of the complexity of the food matrix and the difficulty of quantifying bacteriocin activities in foods, in vitro studies can be performed to simulate and study the in situ functionality of bacteriocinogenic starters. In situ bacteriocin production is most promising for a fast, widespread, and legal use of bacteriocins to achieve the desirable fermentation and a safe final product. The bacteriocin production may be of utmost importance when bacteriocin‐producing LAB are added to foods as starters or protective cultures (adjunct culture). In the current review, our interest is mainly focused on the research of in situ bacteriocin production through finding the potential of the bacteriocinogenic cultures, which have biotechnological importance for the dairy industry.

Use of whey ultrafiltrate as a substrate for production of carotenoids by the yeast Rhodotorula rubra.

Carotenogenesis of the lactose-negative yeast Rhodotorula rubra GED5 was studied by cocultivation with Kluyveromyces lactis MP11 in whey ultrafiltrate (WU) (35, 50, and 70 g of lactose/L). Maximum yields of cell mass (24.3 g/L) and carotenoids (10.2 mg/L of culture fluid or 0.421 µ g/g of dry cells) were obtained by growing the microbial association in WU (50 g of lactose/L) in a fermentor with an airflow rate of 0.8 L/(L·min), agitation of 220 rpm, and temperature of 30°C. The identified carotenoid pigments—β-carotene, torulene, and torularhodin—reached maximum concentrations (133, 26.9, and 222.3 µg/g of dry cells, respectively) on d 5 for torulene and d 6 for β-carotene and torularhodin.

Carotenoid production by lactoso-negative yeasts co-cultivated with lactic acid bacteria in whey ultrafiltrate.

Two strains were selected - the lactoso-negative yeast Rhodotorula rubra GED2 and the homofermentative Lactobacillus casei subsp. casei Ha1 for co-cultivation in cheese whey ultrafiltrate (WU) and active synthesis of carotenoids. Under conditions of intensive aeration (1.0 l/1 min, 220 rpm), a temperature of 30 °C, WU with 55.0 g lactose/l, initial pH = 5.5, the carotenoid content in the cells reached a maximum, when the growth of the cultures had come to an end, i.e. in the stationary phase of the yeast. The maxima for dry cell accumulation (27.0 g/l) and carotenoid formation (12.1 mg/l culture medium) did not coincide on the 5th and 6th day, respectively. A peculiarity of the carotenoid-synthesizing Rh. rubra GED2 strain, co-cultivated with L. casei Ha1, was the production of carotenoids with high β-carotene content (46.6% of total carotenoids) and 10.7% and 36.9% for torulene and torularhodin, respectively.

Effect of oxygen on batch yogurt cultures

A yogurt culture (Streptococcus thermophilus 15HA + Lactobacillus delbrueckii subsp. bulgaricus 2-11) was studied in conditions of aerobic batch fermentation (10–40% dissolved oxygen in milk). The growth and acidification of S. thermophilus 15HA were stimulated at 20% oxygen concentration and the lactic acid process in a mixed culture was shortened by 1 h (2.5 h for the aerobic culture and 3.5 h for the anaerobic mixed culture). Streptococcus thermophilus 15HA oxygen tolerance was significantly impaired at oxygen concentrations in the milk above 30%. Though S. thermophilus 15HA was able to overcome to some extent the impact of high oxygen concentration (40%), the lactic acid produced was insufficient to coagulate the milk casein (4.0 g lactic acid l−1 in the mixed culture and 3.8 g lactic acid l−1 in the pure culture). A dramatic decrease in the viable cell count of L. delbrueckii subsp. bulgaricus 2-11 in the pure and mixed cultures was recorded at 30% dissolved oxygen.

Exopolysaccharides produced by mixed culture of yeast Rhodotorula rubra GED10 and yogurt bacteria (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11)

Aims:  The studies of the production of exopolysaccharides by lactose‐negative yeast and a yogurt starter co‐cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high‐efficiency synthesis of biopolymers by mixed cultivation.

Improvement of Carotenoid-Synthesizing Yeast Rhodotorula rubra by Chemical Mutagenesis

A mutant Rhodotorula rubra with enhanced carotenoid-synthesizing activity for synthesizing total carotenoids and β-carotene was obtained by N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. When co-cultivated with yogurt starter bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus) in whey ultrafiltrate it produced 15.7 mg total carotenoids l-1 culture fluid or 946 μg total carotenoids g-1 dry cells of which 71% was β-carotene. Grown as a monoculture in glucose substrate, the mutant shown 1.4 times lower carotenoid-synthesizing activity, and the relative share of β-carotene in the total carotenoids was lower (63%). The individual pigments torulene and torularhodin were identified, whose mass fractions were (29% and 7%) and (24% and 4%), respectively, for the mutant grown as a monoculture and as a mixed culture with the yogurt bacteria.